RNAi in Zebrafish - vector for zebrafish knock-down (Mar/26/2009 )

Hi,
I am looking for a shRNA vector which can be used in zebrafish. We want to inject the vectors into the zygote to induce a silencing effect in all cells of the organism lasting until adult. Does anyone know such a vector?

We do not want to use Morpholinos as we would like to see effects in the adult fish.

Has anyone used the gene gun as delivery method in zebrafish zygote.

Thanks a lot,
Frank

-Frank Gerlach-

Hi Frank,

A Vivo-Morpholino can be administered to a grown fish. While a Morpholino cannot be expressed in a fish as an siRNA can, you can still use Morpholinos for later stage knockdowns as long as a delivery moiety is attached to the oligo so that it can enter cells from the blood.

Best wishes,

- Jon

-Jon Moulton-

Hi Jon,

thanks. We think this might be problematic, as our gene of interest is exclusively expressed in the brain. Do the vivo-morpholinos cross the blood-brain-barrier? Do you experience with that? How much would you inject and how would you apply the vivo-morpholino? Just by injection of a defined amount into the abdomen?

Thanks and best wishes,
Frank

Jon Moulton on Mar 30 2009, 03:49 PM said:

Hi Frank,

A Vivo-Morpholino can be administered to a grown fish. While a Morpholino cannot be expressed in a fish as an siRNA can, you can still use Morpholinos for later stage knockdowns as long as a delivery moiety is attached to the oligo so that it can enter cells from the blood.

Best wishes,

- Jon

-Frank Gerlach-

Frank Gerlach on Apr 1 2009, 08:34 AM said:

We think this might be problematic, as our gene of interest is exclusively expressed in the brain. Do the vivo-morpholinos cross the blood-brain-barrier? Do you experience with that? How much would you inject and how would you apply the vivo-morpholino? Just by injection of a defined amount into the abdomen?

You're right Frank, that is a tough problem. We know from mouse studies that the Vivo-Morpholinos don't appreciably cross the blood-brain barrier after intravenous injection. In mice, this will likely be solved by intracerebroventricular infusion, but I've never heard of cerebroventricular cannulation of a zebrafish. Could oligo be injected into the brain? Folks have been discussing that approach with us for mouse studies. I think that would be quite a challenge in a zebrafish! So far, no work has been published describing brain knockdowns with Vivo-Morpholinos. I've not addressed your dose questions because clearly there is a bigger roadblock, but here's a start -- in general we look for about 12 mg/kg dose, perhaps this should be scaled to the organ mass for brain injection. Following are a few descriptions of bare Morpholinos administered to rodent brains, but you'd have some experimental hurdles to cross before getting a Morpholino knockdown in Danio. Sorry, I wish the news was better.